Project Summary/Abstract Adoptive therapy involving the ex vivo isolation and expansion of antigen-specific T cells yields the promise of tumor eradication and immunoprotection with minimal toxicity. In recent years, this modality has produced promising results for the treatment of patients with metastatic melanoma. However, factors contributing to the in vivo survival and function of transferred T cells and ultimately, tumor regression in patients, have yet to be defined and optimized. It is believed that pre-infusion conditioning and post-infusion cytokine administration contribute to the efficacy of adoptively transferred T cells. We postulate that the use of T cell clones of defined magnitude, phenotype and specificity for adoptive therapy will permit a precise and rigorous examination of the influence of immunomodulatory regimens and facilitate the identification of elements responsible for a successful strategy. We propose to identify in a Phase I, dose-finding study, a cyclophosphamide conditioning regimen pre- infusion and a dose of IL-2 post-infusion that is safe and that supports the in vivo survival and function of adoptively transferred T cells. In the course of performing this trial we observed T cell persistence of significant duration (from 30 to 300 days) accompanied by RECIST-criteria partial and complete responses. As a dose escalation study, the study was initially designed with a limited number of patients (n=6). In light of these findings, we propose to extend this study to include an additional 6 patients and to more rigorously evaluate the differentiation phenotype of adoptively transferred T cells giving rise to these highly persistent populations in vivo. In response to Notice Number (NOT-OD-09-058) " NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications," we propose to evaluate the genetic and phenotypic signature of these highly persistent adoptively transferred T cell clones in the hope that such T cells can be prospectively identified and enriched for adoptive cellular therapy. PUBLIC HEALTH RELEVANCE: Relevance of Research to Public Health (lay language): Cancers that are resistant to standard chemotherapy and radiation may be treatable using components of the immune system. We propose to use immune cells, T cells, that recognize targets on tumor cells as a means of treating patients with advanced (metastatic) melanoma. By isolating and expanding such T cells and infusing them into patients, we can track the survival and function of these cells and, we hope, identify a treatment approach that will be safe and will improve the effectiveness of melanoma-specific T cells.